ABSTRACT: To detect the relationship between ambient temperature and otolith stable oxygen isotope (δ18O), Pacific bluefin tuna Thunnus orientalis larvae were reared at 6 different temperatures (between 23 and 28°C at 35 psu; mean δ18O value, +0.31‰) for 2 to 8 d after hatching. For the first time, otolith δ18O was measured using a continuous-flow isotope ratio mass spectrometry system. The linear relationship between otolith δ18O and temperature was determined as follows: δotolith – δwater = –2.70 (T°C) + 5.193 (r2 = 0.806, p < 0.01). The temperature-dependent fractionation of otolith δ18O was close to that reported for inorganic aragonite, indicating that the vital effects of isotopes are small. The otolith δ18O values of juveniles reared at a mean of 26.5°C were not significantly different from those of larvae reared at 26°C. With regard to the 2 pairs of otoliths within an individual fish, the differences in δ18O values between the left and right otoliths were subtle. The otolith δ18O values of larvae reared in δ18O-depleted seawater (24 to 26°C at 32 psu; δ18O, –0.17‰) were lower, but were not significantly different from those of larvae reared in +0.31‰ water, which is the δ18O value of the water in which the spawners were raised. These results suggest that the sample size for the 32 psu experiment was too small and that the rearing durations were too short to affect the otoliths completely. Our results demonstrate that otolith δ18O values of Pacific bluefin tuna larvae can provide precise and accurate estimates of their ambient temperature experience.